Solution and method for adhering suspension components to a substrate

ABSTRACT

This disclosure is directed to a method and an attachment solution for adhering a cytological or histological sample, such as buffy coat, to a substrate, such as a microscope slide. The attachment solution includes an attachment base, an anti-coagulant, and a nonsteroidal anti-inflammatory drug. The attachment base may be an alcohol, an acid, an oxidizer, an organohalogen, a ketone, or any combination thereof. Once a sample is obtained, the sample may be re-suspended in the attachment solution or the attachment solution may be added to the sample. The sample may then be dispensed onto an analysis platform as one or more droplets and cured.

CROSS-REFERENCE TO A RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.14/956,622, filed Dec. 2, 2015.

TECHNICAL FIELD

This disclosure relates generally to analyzing a suspension and, inparticular, to a solution and method for adhering a component of asuspension to a substrate.

BACKGROUND

Current diagnostic methods and techniques rely on the interpretation ofhistological and cytological samples. It is an important aspect in theseinterpretations that the sample remains adhered to a substrate on whichthe samples are being analyzed and processed, such as a microscopeslide. Valuable diagnostic information may then be obtained from thesample upon subsequent processing due to the adherence of the sample.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flowchart of an example method for making an exampleattachment solution.

FIG. 2 shows a flowchart of an example method for adhering a sample to asubstrate.

FIG. 3A shows an example sample having been re-suspended in an exampleattachment solution.

FIG. 3B shows the example sample mixing with the attach solution to forma re-suspended cytological sample.

FIG. 3C shows the re-suspended sample being deposited on an exampleanalysis platform.

FIG. 3D shows the re-suspended sample having been spread on and adheredto the analysis platform.

DETAILED DESCRIPTION

This disclosure is directed to a method and an attachment solution foradhering a cytological or histological sample to a substrate. Theattachment solution includes an attachment base and a nonsteroidalanti-inflammatory drug. The attachment base may be an alcohol, an acid,an oxidizer, an organohalogen, a ketone, such as acetone, or anycombination thereof, such as Carnoy's solution. The attachment solutionmay also include an anti-coagulant. Once a sample is obtained, thesample may be re-suspended in the attachment solution or the attachmentsolution may be added to the sample. The sample is dispensed onto ananalysis platform as one or more droplets and cured. The sample may thenbe fixed, permeabilized, labeled, blocked, and washed. The sample maythen be imaged and analyzed.

In the following description, the term “sample” is used to describe aspecimen to be analyzed. The specimen may be a suspension, a portion ofthe suspension, or a component of the suspension. For example, when thesuspension is anticoagulated whole blood, the sample may be theanticoagulated whole blood (i.e. a suspension), the buffy coat (i.e. aportion of the suspension), or a circulating tumor cell (i.e. acomponent of the suspension).

Attachment Solution

The attachment solution may adhere a sample to a substrate. The samplecan be a buffy coat of a blood sample and the substrate can be thesurface of a microscope slide. The attachment solution includes anattachment base and a nonsteroidal anti-inflammatory drug. Theattachment solution may also include water. A buffer may also beincluded or a buffered solution may be used in place of or in additionto the water. A buffer or a buffer of the buffered solution may include,but is not limited to, ACES, ADA, AMP, AMPD, AMPSO, BES, Bicine,BIS-TRIS, BIS-TRIS propane, CABS, CAPS, CAPSO, CHES, DIPSO, EDTA, HEPPS(EPPS), Gly-Gly, HEPBS, HEPES, MES, MOPS, MOPSO, PIPES, POPSO, potassiumphosphate, sodium phosphate (monobasic and/or dibasic), TAPS, TAPSO,TES, Tricine, tris, Trizma®, and combinations thereof. The pH of thebuffer or buffer solution may range from 5 to 12.

It should be noted that in a first embodiment, the attachment solutionincludes a salt, such as that in a saline solution, where the attachmentsolution remains hypotonic relative to the sample, as an isotonic orhypertonic attachment solution may cause the sample to crack whiledrying or curing. Alternatively, the attachment solution does notinclude a salt, such as that included in a saline solution, as the saltmay cause the sample to crack while drying or curing.

The attachment base may include an alcohol, such as ethanol, methanol,propanol, isopropanol, butanol, and an acid, an oxidizer, anorganohalogen, a ketone, such as acetone, or any combination thereof,such as Carnoy's solution. The attachment base may be capable of fixingsample components, such as cells, without cross-linking other samplecomponents, such as proteins. Preventing cross-linking of the othersample components, such as proteins, may avoid an additional processingstep, such as antigen retrieval. In other words, fixing samplecomponents to a substrate with the attachment base eliminates theprocess of antigen retrieval. The attachment base may also have a fast(i.e. less than one hour) cure time. The attachment base may have afinal concentration, such as when the attachment solution and the samplehave been mixed, of 60-70% by volume, and more specifically 63-68% byvolume. Having a concentration of the attachment base outside thedesired range may affect the sample or a portion thereof, such as bylysis, clumping, inability to adhere to the substrate, or the like. Thefinal concentration of the attachment base may be determined by ameasured property of the sample to be adhered including, but not limitedto, sample volume, packed cell volume, hematocrit level, blood type,sample type (i.e. blood, urine, buffy coat, single cell, etc.), or thelike.

The nonsteroidal anti-inflammatory drug may be, but is not limited to,acetylsalicylic acid, celecoxib (Celebrex), diclofenac (Voltaren),diflunisal (Dolobid), etodolac (Lodine), ibuprofen (Motrin),indomethacin (Indocin), ketoprofen (Orudis), ketorolac (Toradol),nabumetone (Relafen), naproxen (Aleve, Naprosyn), oxaprozin (Daypro),piroxicam (Feldene), salsalate (Amigesic), sulindac (Clinoril), tolmetin(Tolectin), or the like. The nonsteroidal anti-inflammatory drug may actas an antithrombotic (i.e. reducing blood clotting, such as by reducingthe blood clotting function of various blood components). Thenonsteroidal anti-inflammatory drug may have a final concentration of100-1000 ug/mL.

The attachment solution may also include an anti-coagulant which may be,but is not limited to, heparin, heparin sodium, heparin/dextrose,ethylene-diamine-tetra-acetic acid, dalteparin sodium, argatroban,bivalirudin, lepirudin, or the like. The anti-coagulant preventscoagulation (i.e. clumping, clotting or solidification) of the sample.The anti-coagulant may have a final concentration of 10-1000 ug/mL.

The attachment solution may also include a biological polymer toincrease the adhesion between the sample and the substrate. Thebiological polymer may include, but is not limited to, biomimeticadhesive polymers (such as polyphenolic protein from marine life;3,4-dihydroxyphenylalanine), fibrin glue,gelatin-resorcinol-formaldehyde, poly-L-lysine, poly-D-lysine, or thelike.

Method of Making Attachment Solution

The following are example reagents used to make attachment solutions:

-   1. Heparin 2000 μg/mL: Prepared by dissolving 20 mg of heparin    sodium salt (Sigma H3393) in 10 mL of water-   2. BD Cell-Tak: BD Cell-Tak® Cell and Tissue Adhesive (BD    354241)—approximately 1500 μg/mL stock concentration-   3. Cell-Tak Buffer 5×: Prepared by dissolving 21 g sodium    bicarbonate (NaHCO₃) into 500 mL of water-   4. Cell-Tak Buffer 1×: Prepared by dissolving 4.2 g sodium    bicarbonate (NaHCO₃) into 500 mL of water and adding 2.5 mL 1M    hydrochloric acid (HCl)-   5. Sigma Aldrich 494437: Methanol (about 99.93% by volume)

The following are example formulations for attachment solutions, as seenin FIG. 1:

-   -   1. 70% Methanol by volume, 100 ug/mL Heparin, 300 ug/mL of        acetylsalicylic acid, 50 ug/mL Cell-Take:        -   A. Mix 350 uL of Cell-Take with 650 uL of Cell-Tak Buffer 1×        -   B. Mix thoroughly        -   C. Add 1.5 mL of water        -   D. Add 500 uL of Heparin 2000 ug/mL        -   E. Add 7.0 mL of Methanol        -   F. Add 3000 ug of acetylsalicylic acid        -   G. Mix thoroughly    -   2. 60% Methanol by volume, 300 ug/mL of acetylsalicylic acid:        -   A. Add 6000 uL of methanol to 4000 uL of water        -   B. Add 3000 ug of acetylsalicylic acid        -   C. Mix thoroughly

Method of Using Attachment Solution

FIG. 2 shows an example method for adhering a sample to a substrate. Inblock 202, a sample is obtained. To obtain the sample, the sample may bewithdrawn directly from a subject or the sample may undergo enrichmentand/or isolation from the suspension. The sample may be enriched by anyappropriate enrichment process including, but not limited to, sequentialdensity fractionation, magnetic-activated cell sorting,fluorescence-activated cell sorting, differential lysis, depletionfilters, or the like. Sequential density fractionation is a process bywhich a suspension is divided into fractions or a fraction of asuspension is divided into sub-fractions by a step-wise or sequentialprocess, such that each step or sequence results in the collection orseparation of a different fraction or sub-fraction from the precedingand successive steps or sequences. The sample may be obtained from othersuspension components by selecting the sample with a device for picking,such as a cell picker, a pipet, a syringe, or the like.

Referring back to FIG. 2, in block 204, the sample 304 is re-suspendedin an attachment solution 306 in a vessel 302, as shown in FIG. 3A.Alternatively, the attachment solution may be added to or mixed with thesample. In block 206, the re-suspended sample 308, which includes atleast a portion of the attachment solution, as shown in FIG. 3B, isdispensed onto or into an analysis platform 310 by a dispenser, such asa pipet or repeating pipet, and spread across the analysis platform. InFIG. 3C, the sample 308 is spread across the analysis platform 310 by aspreader 312, such as a squeegee, a pipet tip, a blade, a two-piecespreader including a blade and a base. Alternatively, the sample 308 maybe spread across the analysis platform 310 by centrifuging, wetting, ornutating the analysis platform 310. The re-suspended sample 308 iscured, as shown in FIG. 3D, to adhere the re-suspended sample 308 to theanalysis platform 310. Alternatively, the re-suspended sample 308 may bedispensed onto the analysis platform 310 and cured without being spreadacross the analysis platform 310. Curing may occur in air, such as atroom temperature; in an environmentally-controlled chamber, such as at37° C.; or the like. Furthermore, the sample may undergo an additionalfixation step, such as in formalin or any appropriate fixative, afterthe curing step has been completed.

The attachment solution may be compatible with any appropriate analysismethod or technique, though more specifically extracellular andintracellular analysis including immunofluorescent labeling and imaging;intracellular protein labeling; chromogenic staining; molecularanalysis; genomic analysis or nucleic acid analysis, including, but notlimited to, genomic sequencing, DNA arrays, expression arrays, proteinarrays, and DNA hybridization arrays; in situ hybridization (“ISH”—atool for analyzing DNA and/or RNA, such as gene copy number changes);polymerase chain reaction (“PCR”); reverse transcription PCR; orbranched DNA (“bDNA”—a tool for analyzing DNA and/or RNA, such as mRNAexpression levels) analysis. Some of the intracellular proteins whichmay be labeled include, but are not limited to, cytokeratin (“CK”),actin, Arp2/3, coronin, dystrophin, FtsZ, myosin, spectrin, tubulin,collagen, cathepsin D, ALDH, PBGD, Akt1, Akt2, c-myc, caspases,survivin, p27^(kip), FOXC2, BRAF, Phospho-Akt1 and 2, Phospho-Erk1/2,Erk1/2, P38 MAPK, Vimentin, ER, PgR, PI3K, pFAK, KRAS, ALKH1, Twist1,Snail1, ZEB1, Fibronectin, Slug, Ki-67, M30, MAGEA3, phosphorylatedreceptor kinases, modified histones, chromatin-associated proteins, andMAGE.

The analysis platform 310 may be a microscope slide, a positivelycharged microscope slide, a coated microscope slide, a porous slide, amicro-well slide, a well plate, a coverslip, a cell microarray, or thelike. The analysis platform 310 may be any appropriate material,including, but not limited to, glass, plastic, ceramic, metal, or thelike.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the disclosure.However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the systems and methodsdescribed herein. The foregoing descriptions of specific embodiments arepresented by way of examples for purposes of illustration anddescription. They are not intended to be exhaustive of or to limit thisdisclosure to the precise forms described. Many modifications andvariations are possible in view of the above teachings. The embodimentsare shown and described in order to best explain the principles of thisdisclosure and practical applications, to thereby enable others skilledin the art to best utilize this disclosure and various embodiments withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of this disclosure be defined by thefollowing claims and their equivalents:

We claim:
 1. A solution consisting essentially of: an alcohol having afinal concentration of 60-70% by volume; acetylsalicyclic acid having afinal concentration of 100-1000 ug/mL; and water, wherein the finalconcentrations are the concentrations of the respective components whenmixed with a sample.
 2. The solution of claim 1, wherein the alcohol ismethanol.
 3. The solution of claim 1, further comprising a buffer. 4.The solution of claim 1, further comprising a salt, wherein the solutionis hypotonic relative to the sample.